Single Cell Atlas Construction Of Bovine Skeletal Muscle Development Based On ScRNA-seq And ScATAC-seq

Qinchuan cattle,as one of the excellent local cattle breeds in China,has the advantages of resistance to rough feeding and excellent meat quality.It belongs to the large-sized dual-purpose of cattle for both draft and beef,and is mainly used as meat production.However,it still has the disadvantages of slow growth and low meat yield.Although it has been improved by the traditional hybridization technique,the improvement progress is very slow.Muscle yield and mass are important economic traits of beef cattle.Longissimus dorsi muscle is a typical skeletal muscle and the source of high-end beef in livestock husbandry.It is the optimal material for studying meat production traits.Skeletal muscle is a complex heterogeneous tissue,and characterizing its cellular heterogeneity and transcriptional and epigenetic signatures are important for understanding the details of its ontogeny.In this study,the longissimus dorsi muscle of Qinchuan cattle was selected as the research object for the development of bovine skeletal muscle,in order to deepen the understanding process of bovine skeletal muscle development,and provide reference for related research on skeletal muscle development.Furthermore,it provides theoretical basis and technical support for precise breeding of bovine.This study applied scRNA-seq and sc ATAC-seq to analyze the cell types,molecular features,transcriptional and epigenetic regulation,and patterns of developing bovine skeletal muscle from gestational,lactational and adult stages.Detailed molecular analyses were used to dissect cellular heterogeneity,and deduced the differentiation trajectory of myogenic cells and uncovered their dynamic gene expression profiles.SCENIC analysis was performed to demonstrate key regulons during cell fate decisions.It explored the future expression states of these heterogeneous cells by RNA velocity analysis and found extensive networks of intercellular communication using the toolkit Cell Chat.Moreover,the transcriptomic and chromatin accessibility modalities were confirmed to be highly concordant,and integrative analysis of chromatin accessibility and gene expression revealed key transcriptional regulators acting during myogenesis.The main results of this study are as follows:1.Identification of skeletal muscle cell heterogeneity in fetal,calf and adult bovineTranscriptomic analysis was performed on skeletal muscle cells in fetal,calf and adult bovine by using scRNA-seq technology.Bovine skeletal muscle cells were divided into 29 clusters through cluster analysis,and identified 18 cell types,such as adipocytes,myoblasts,and myocytes,as well as eight skeletal muscle myogenic subgroups.These include myoblasts,myocytes,myogenic progenitors,Type I fibers 1,Type I fibers 2,type IIA fibers1,type IIA fibers 2,and type IIX fibers.The significant changes of cell components were detected in three different developmental stages.Fibroblasts accounted for the largest proportion in fetal bovine.In calf bovine,endothelial cells accounted for the most,followed by type IIX fibers.In adult bovine,type IIX fibers make up the largest proportion.2.Construction of single cell transcriptome atlas for skeletal muscle in fetal,calf and adult bovineThe pseudotime trajectory exhibited a distinct sequential ordering for these myogenic subpopulations and eight distinct gene clusters were observed according to their expression pattern.The expression patterns of these 1493 genes showed that the expression of some genes showed a monotonic trend in the pseudotime,while others showed non-monotonic behavior.For example,most of gene cluster 1,such as TPM3,TNNT1,MYH7,CSRP3,MUSTN1,and NMRK2,are expressed monotonically and incremically in pseudotime.MEGF10,SORCS3,IGFBP6,HMCN2 and SASH1 in gene clusters 2,4,5,6 and 8 showed high levels of non-monotony gene expression.KEGG enrichment analysis showed that eight different gene clusters of the skeletal muscle myogenic subset were found in pseudotime,which were mainly regulated by calcium signaling pathway,PI3K-AKT signaling pathway and Fox O signaling pathway.RNA velocity analysis was used to explore the likely future transcriptional states of individual cells and the developmental dynamics of differentiation among neighbouring cells.Found that Type II fibres are the majority of cells at the adult stage,and RNA velocity vectors of Type I fibres 2 point towards Type IIA fibres 1.Moreover,Type IIA fibres 1 and Type IIA fibres 2 have RNA velocity vectors pointing towards the Type IIX fibres.The single cell transcription atlas of bovine skeletal muscle was constructed.SCENIC regulatory inference analysis was applied to identify specific transcriptional regulators associated with muscle development,and it is predicted that MSC,MYF5,MYOD1,FOXP3,ESRRA,BACH1,SIX2 and ATF4 might actively regulate the transcription of myogenic progenitors,myoblasts,myocytes,type I fibers 1,type I fibers 2,type IIA fibers 1,type IIA fibers 2 and type IIX fibers,respectively.Cell Chat analysis on the scRNA-seq data set then classified many ligand–receptor pairs among these cell clusters,which were further categorized into significant signalling pathways,including BMP,IGF,WNT,MSTN,ANGPTL,TGFB,TNF,VEGF and FGF.3.Construction of single cell chromatin accessibility atlas of skeletal muscle in fetal,calf and adult bovineChromatin accessibility analysis of skeletal muscle cells in fetal,calf and adult bovine was performed by using sc ATAC-seq technology.Bovine skeletal muscle cells were identified 11 cell types,such as adipocytes,endothelial cells,lymphocytes,myoblasts,and type IIX fibers,as well as five skeletal muscle myogenic subgroups.These include myoblasts,myocytes,myogenic progenitors,Type I fibers 1,Type I fibers 2,type IIA fibers1,type IIA fibers 2,and type IIX fibers.The significant changes of cell components were detected in three different developmental stages.Fibroblasts accounted for the largest proportion in fetal bovine.In calf and adult bovine,type IIX fibers occupy the largest proportion.By evaluating the expression trend of myogenic genes within the pseudotime,trajectory inference is highly consistent with scRNA-seq.There was no expression of PAX7,ACTC1 and MYF5 in the final stage of the pseudotime,and no expression of MYOD1 within the pseudotime.This research revealed the accessibility dynamics of five muscle derived subpopulations and constructed a single cell chromatin atlas of bovine skeletal muscle.Known and de novo transcription factors of 11 cell types were predicted by motifs enrichment analysis,and a series of specifically expressed transcription factors was revealed,such as MYF5,MYOG,SIX2,TCF12,ZBTB18 and FOSL2.4.Combined analysis of scRNA-seq and sc ATAC-seqscRNA-seq and sc ATAC-seq results were successfully integrated,which confirmed that the two models were highly consistent in cell heterogeneity and gene expression patterns.Three significantly expressed intersection genes EPHA3,NES and ENSBTAG00000054182,and significantly shared transcription factors were found,such as MYOG,SIX2,MEF2 C,MEF2D,MITF,TEAD4 and ESRRB,that are likely to be candidates for the promotion of cell fate transition during bovine skeletal muscle development.Immunohistochemistry was used to confirm the differential expression of PAX7,ACTC1,MYH1,MYOG and COL3A1 at different developmental stages in bovine skeletal muscle,as well as the specific transcriptional regulation of transcription factors SIX2,FOXP3,ATF4,POLE3,RCOR1,HLF and WT1.The authenticity of the sequencing analysis results was further confirmed by RT-q PCR.In conclusion,we constructed a single-cell dynamic chromatin/transcriptional atlas for normal bovine skeletal muscle development based on scRNA-seq and sc ATAC-seq.These provide new insights into understanding the structure and function of mammalian skeletal muscle,which will provide powerful theoretical and technical support for cattle breeding research,and promoting the development of the cattle industry.